The therapeutic potential of human embryonic stem cells is extraordinary. Without a doubt, regenerative medicines will save thousands of lives in the years to come. Before that day arrives, much needs to be learned from the cells themselves. The reasons that these cells hold so much promise are two-fold: (1) embryonic stem cells can renew themselves indefinitely (divide and divide and…) and (2) embryonic stem cells can be trained to become any cell type of the body (neurons, heart muscle, skin, liver, kidney…). However, it should be emphasized that these two points are only valid if the growth conditions are properly established. While we have made great strides in developing culture conditions that can support self-renewal of embryonic stem cells, we are a long way from mastering the conditions necessary for differentiating embryonic stem cells into every cell type of the body (of which there are about 200). Ultimately, if therapies based on stem cells are to be realized, these cells will have to be grown in massive quantities, with an unprecedented level of quality control to ensure that only one cell type can be found in the lot. Furthermore, the fate of stem cells is crucial to their use in new therapies—in other words, these cells must be kept alive and functional to have benefit to human patients.

However, one of the major challenges facing the growth of embryonic stem cells is the abundance of cell death that occurs. Cells typically die when their needs are not met (either lack of proper nutrients or growth factors) or when they face harsh conditions. If we could somehow block the cell death that occurs in these cultures or if we could change the conditions to remove the components that trigger cell death, we could achieve growth of hESCs of a greater scale. It turns out that when cells die, they do not do so passively. Instead, once given a “go” signal, cells utilize their own energy and cellular machinery to dismantle themselves, a process known as programmed cell death. There are at least five major forms of programmed cell death: apoptosis (the best described pathway), autophagic cell death, PARP-mediated cell death, paraptosis, and calcium-mediated programmed cell death. Each of these programmed cell death pathways are activated by different stresses. In the proposed research, we aim to determine which of the five major forms of programmed cell death occur in hESCs. Furthermore, we will evaluate how the repertoire of PCD pathways changes when hESCs change, or differentiate, into neurons. At the same time that we will be learning about the most appropriate conditions for growing hESCs, we will also be able to determine which conditions are ideal for cultivating neurons, which could ultimately be used in regenerative medicine therapies.

Statement of Benefit to California:

In passing Proposition 71, Californians have ushered in a new era of human embryonic stem cell research. However, before the therapeutic potential of human embryonic stem cells can be realized, several key issues relevant to programmed stem cell death must be addressed: (1) we must understand what insults trigger PCD in stem cells; (2) we must understand what programs of cell death are available to stem cells and stem cell-derived differentiated cells; (3) we must understand how to block cell death induction in hESCs and their derivatives; (4) we must address the technical hurdles of propagating these cells at an industrial scale. The goal of our research is to determine what measures might be taken to permit such a wide-scale expansion effort. Our laboratory has constructed a mechanistic taxonomy of cell death programs, and therefore has a unique ability to identify various novel forms of cell death that may occur in hESCs. Because improved methods of human embryonic stem cell propagation will stimulate research on human embryonic stem cells, the benefit of our research to Californians will be seen repeatedly and, ultimately, in the delivery of human embryonic stem cell-based therapies

Progress Report:

Our CIRM SEED grant proposal was to study the pathways of programmed cell death (cell suicide) in human embryonic stem cells. This is a critical area for several reasons: for example, when we transplant stem cells, we need to know how to keep them from dying so that they can be functional. On the other hand, we also need to know how to induce programmed cell death in stem cells, since it is becoming more and more clear that cancers may be propagated by stem cell populations. For these and many other reasons, it is important to know what pathways of programmed cell death are available to stem cells.

There are at least five major forms of programmed cell death: apoptosis (the best described pathway), autophagic cell death, PARP-mediated cell death, paraptosis, and calcium-mediated programmed cell death. Each of these programmed cell death pathways is activated by different stimuli and stresses. In the proposed research, we aimed to determine which of the five major forms of programmed cell death occur in human embryonic stem cells (hESCsP). Furthermore, we evaluated how the repertoire of PCD pathways changes when hESCs differentiate into neurons.

We first compiled a list of 322 genes whose activity contributes to these various forms of programmed cell death. Of these 322 genes, 311 were found to be represented on the assay system we used. 153 of these genes were measured with a very high detection confidence (0.95 or greater). We performed a special analysis (unsupervised two-way hierarchical cluster analysis) of these genes and represented the expression profiles in a heat-map. Within this group of genes, we chose to focus our attention first on Bcl-2 family members (both pro-apoptotic and anti-apoptotic) because we found transcripts of these gene families to be some of the most differentially expressed within the 43 samples analyzed. We also focused on this gene family because it is a critical family for the control of programmed cell death.

We then quantified all members of the Bcl-2 family amongst hESCs and differentiated cells, working under the hypothesis that overly abundant Bcl-2 family member transcripts in hESCs would point toward apoptotic and/or anti-apoptotic signaling cascades that are especially active in hESCs. We were encouraged when we found that the expression of some Bcl-2 family member genes changed dramatically (some up and others down) when hESCs were differentiated to other cell types.

We found that apoptosis is readily activated in hESCs, and, surprisingly, that a subset of p53-induced Bcl-2 family genes (e.g., Noxa and Puma) is highly constitutively expressed in hESCs (in comparison to multiple non-stem-cell primary cells). Whereas the pro-apoptotic genes Noxa and Puma are typically expressed only in response to DNA damage and p53 activity, hESCs constitutively express high levels of Noxa and Puma. This finding suggests that embryonic stem cells might be hyper-sensitive to sources of DNA damage like ultraviolet rays and X-irradiation, compared to other cell types, and furthermore, that p53-independent mechanisms of death induced by DNA damage might be operative in hESCs. However, not all p53-induced genes are up-regulated in these cells, since p21 is not up-regulated. These findings raise the important possibility that cultured hESCs may undergo DNA damage despite appropriate culture conditions, which would be a critical issue for hESC growth for transplantation. Another possibility is that p53, the “guardian of the genome”, is indeed protecting hESCs from DNA damage, in part by having a low threshold to activate programmed cell death, but without activating senescence (since p21 was not found to be up-regulated). Thus p53 may, in hESCs, mediate hypersensitivity to DNA damage, as a mechanism to keep the genomes of hESCs “pristine” for long-term functionality. We are performing follow-up studies to determine the mechanism and implications of the striking constitutive up-regulation of this subset of p53 target genes.

We are grateful to CIRM for supporting this SEED grant, especially since it has allowed us to identify novel aspects of programmed cell death and the underlying molecules, and to identify a potentially important novel aspect of human embryonic stem cells that may prove to be important in the consideration of transplantation of these cells and their differentiated derivatives.